Antigen-specific immunotherapy is an essential component of strategies for prevention and intervention in type 1 diabetes. The combination of an excellent safety profile and the potential for restoration of -cell specific tolerance is the major appeal of this approach and place antigen-specific immunotherapy in a potentially key position among the currently known therapeutic options. However, many questions remain in relation to the optimal choice of antigen, the mode and timing of delivery, dosing, and the composition of the drug. We also need to discover the best biomarkers for monitoring the efficacy of such therapy. To address these gaps in knowledge requires mechanistic studies, preferably conducted in humans and in the context of an ASI clinical trial. These studies have the potential to identify new monitoring technologies and reveal immune alterations which, when combined, assist in further drug development. This proposal is centred on the TrialNet study of Diamyd GAD65-alum administration to patients with new- onset type 1 diabetes. Our preliminary data from this pilot study demonstrate an increased GAD65-specific T cell response in the vaccinated subjects but not in the placebo group. These findings further indicate that GAD65-alum administration induces T cell response targeted to pre-existing and novel target epitopes, which may be regulatory. The proposed study will provide new knowledge for future antigen-specific therapies in type 1 diabetes through (i) discovery of boosted autoantigen responses (pre-existing memory and de novo epitopes); (ii) Identification of previously unknown, therapy-related targets, which induce reactivity with a potentially protective function; (iii) development f reagents in tetramer or ELISPOT platforms as novel biomarkers that are useful in the antigen specific therapies; (iv) characterization of the functional nature and the immunologic profile of the drug induced T cell responses; and (v) improved understanding of antigen- presenting cell types required for these responses. PUBLIC HEALTH RELEVANCE: This research will generate knowledge that is of utility in maximizing the potential of antigen specific immunotherapy for autoimmune disease, as well as furthering our understanding of how autoimmunity arises.